The SmMYB36-SmERF6/SmERF115 module regulates the biosynthesis of tanshinones and phenolic acids in salvia miltiorrhiza hairy roots

Abstract Tanshinone and phenolic acids are the most important active substances of Salvia miltiorrhiza, and the insight into their transcriptional regulatory mechanisms is an essential process to increase their content in vivo. SmMYB36 has been found to have important regulatory functions in the synthesis of tanshinone and phenolic acid; paradoxically, its mechanism of action in S. miltiorrhiza is not clear. Here, we demonstrated that SmMYB36 functions as a promoter of tanshinones accumulation and a suppressor of phenolic acids through the generation of SmMYB36 overexpressed and chimeric SmMYB36-SRDX (EAR repressive domain) repressor hairy roots in combination with transcriptomic-metabolomic analysis. SmMYB36 directly down-regulate the key enzyme gene of primary metabolism, SmGAPC, up-regulate the tanshinones biosynthesis branch genes SmDXS2, SmGGPPS1, SmCPS1 and down-regulate the phenolic acids biosynthesis branch enzyme gene, SmRAS. Meanwhile, SmERF6, a positive regulator of tanshinone synthesis activating SmCPS1, was up-regulated and SmERF115, a positive regulator of phenolic acid biosynthesis activating SmRAS, was down-regulated. Furthermore, the seven acidic amino acids at the C-terminus of SmMYB36 are required for both self-activating domain and activation of target gene expression. As a consequence, this study contributes to reveal the potential relevance of transcription factors synergistically regulating the biosynthesis of tanshinone and phenolic acid.


Introduction
The different phenylpropanoid-derived compounds have a multitude of biological functions, including pigments, cell wall components, UV-absorbing compounds, phytoalexins, and modulators of developmental signaling. The MYB family of proteins is one of the largest transcription factor families and is regulator of phenylpropanoid synthesis in plants. Most subgroup 5 R2R3-MYB proteins are responsible for proanthocyanidin synthsis, most subgroup 6 R2R3-MYB proteins take part in anthocyanin synthesis, while the majority of R2R3-MYB subgroup 7 proteins contribute to f lavonol synthesis. Most 1R-MYB and subgroup 4 R2R3-MYB proteins tend to be negative regulators of phenylpropanoid synthesis. MYB-bHLH-WD40 (MBW) complexes containing R2R3-MYB (subgroup 5 and 6), bHLH and WD40-repeat proteins regulate anthocyanin and proanthocyanidin synthsis [1,2].
In this study, we elucidated the regulatory functions of SmMYB36 on tanshinone and phenolic acid biosynthesis more deeply by generating SmMYB36 overexpression and chimeric SmMYB36-SRDX repressor hairy roots and combining them with transcriptome-metabolome analysis. SmMYB36 was found to promote tanshinone synthesis and inhibit phenolic acid synthesis in three main regulatory ways: balance of primary and secondary metabolism; direct and indirect combination; and simultaneous positive and negative regulation. Additionally, the acidic amino acids of SmMYB36 154-160 are required for the structural domains of transcriptional activation, also for the activation of target genes. Taken together, these results show the specific diversity, complexity, and regulatory gene expression network of SmMYB36 regulation of tanshinones and phenolic acids.

SmMYB36 promoted the accumulation of tanshinone and inhibited the accumulation of phenolic acid in the hairy roots of S. miltiorrhiza
To investigate the role of SmMYB36 in the synthesis of tanshinone and phenolic acid, pGWB18-Myc-SmMYB36 (M36O) and pK7WG2R-SmMYB36-SRDX (36R) were transformed into hairy roots ( Fig. S1a and b, see online supplementary material). The extracts of the M36O positive lines have a darker color than the control, while the 36R positive lines are essentially the same as the control (Fig. S1c, see online supplementary material). For the functional analysis of 36R hairy roots,the qRT-PCR results showed that SmMYB36 expression in the three positive lines (36R1, 36R2, and 36R3) was 1.96-fold, 3.11-fold, and 3.77-fold higher than the control, respectively (Fig. S2, see online supplementary material). Compared with the control, the 36R positive lines showed significantly higher RA, Sal B, and TSA contents (P < 0.05), with an average increase of at least 1.6-fold, 1.5-fold, and 1.3-fold in RA, Sal B, and TSA contents (Fig. 1a). In contrast, the tanshinone components DTI, TA1, TAIIA, and TTA were significantly decreased in the 36R lines compared to the control (P < 0.05), while the CT content did not change significantly. Furthermore, as shown in Fig. 1c, phenolic acid biosynthesis pathway genes SmPAL1 and SmRAS were up-regulated 2.12-5.27fold, SmCYP98A14 was down-regulated about 0.45-0.59-fold, and tanshinone biosynthesis pathway genes SmCPS1, SmKSL1, and SmCYP76AH1 were down-regulated 0.31-0.77-fold, respectively, compared with the control.
For the functional analysis of M36O hairy root, qRT-PCR analysis revealed that SmMYB36 expression was 2.5-, 3.5-, and 7.3fold higher in the overexpression lines M36O1, M36O2, and M36O3 than the control (Fig. S2, see online supplementary material). Compared with the control lines, the content of RA, SalB, and TSA in the M36O lines was significantly lower (P < 0.05), and all could be decreased by up to more than 50% (Fig. 1b). The expression of SmPAL1 and SmRAS, key genes of the phenolic acid biosynthesis pathway, decreased approximately 36%-57%, whereas SmCYP98A14 showed no significant difference compared to the control. In addition, the tanshinone biosynthesis pathway genes SmCPS1, SmKSL1, and SmCYP76AH1 were increased by around 1.90-18.77-fold, 3.26-6.35-fold, and 20.92-26.77-fold, respectively (Fig. 1d). Consequently, these results suggest that SmMYB36 functions as a positive regulator of tanshinone biosynthesis a negative regulator of phenolic acid biosynthesis.

Overexpressing SmMYB36 altered different primary and secondary metabolites by widely targeted metabolic profiling
To investigate the metabolic changes in the SmMYB36 overexpression line compared with the control line, widely targeted metabolome was applied. A total of 327 metabolites were detected, including carbohydrates, amino acid and derivatives, mucleotide and derivates, vitamins and derivatives, organic acids and derivative, lipids, alcohols, terpenes, phenolamides, quinones, alkaloids, indole derivatives, sterides, phenolamides, phenylpropanoids, anthocyanin, f lavone, f lavonoid, f lavonol, isof lavone, polyphenol, et al. (Table S3, see online supplementary material).
Then, comparing the SmMYB36 overexpression line with control line, there were 142 differential metabolites, of which 109 were down-regulated and 33 were up-regulated (Table S4, see online supplementary material). In this study, 26 differential amino acid and derivatives (23 down and 3 up) were identified, specially including nine down-regulated standard amino acds (L-histidine, L-glutamine, L-proline, L-asparagine, L-phenylalanine, L-alanine, L-tyrosine, L-leucine, L-isoleucine). Succinic acid in TCA cycle was down-regulated. Twenty-four differential phenylpropanoid metabolites (21 down and 3 up) were identified, including 2 anthocyanins (2 down), 2 f lavones (1 down and 1 up), 3 f lavonols (3 down), 2 isof lavones (1 down and 1 up), 1 polyphenol (1 down), and 14 unclassified phenylpropanoids (13 down and 1 up). In view of the fact that the content of RA and SalB was significantly reduced in SmMYB36 transgenic lines, overexpression of SmMYB36 decreased phenylpropanoid production in S. miltiorrhiza. Four differential terpenoid metabolites were identified, including one monoterpenoid (geniposidic acid), two sesquiterpenoids (roseoside, β-caryophyllene), one diterpenoid (phytol), of which all were down-regulated. Considering that the content of DTI, CT, TA1, and TAIIA was also significantly increased in 36O lines, overexpression of SmMYB36 increased diterpenoid yield in S. miltiorrhiza. As a result, these results also suggest that SmMYB36 up-regulates tanshinone content and down-regulates phenolic acid content by inf luencing the synthesis of various primary and secondary metabolites.
Overexpressing SmMYB36 altered different primary and secondary metabolite parthway biosynthesis enzyme gene by transcriptome profiling To better analyse the potential roles of SmMYB36 in primary and secondary metabolic regulation, we conducted transcriptome profiling for the SmMYB36 overexpression line compared with the control line. Compared to control lines, a total of 6726 DEGs were detected in the SmMYB36 overexpression line, which was comprised of 3583 genes up-regulated and 3143 genes downregulated. All the DEGs were annotated according to GO, KEGG, KOG, Nr, Swiss-Prot, and TrEMBL (Table  S5, see online supplementary material). These DEGs were assigned to three classes of GO: molecular functions, cellular compartment, and biological process. The GO terms 'cellular process', 'metabolic process' and 'response to stimulus' are the most highly enriched among biological process (Fig. S3a, see online supplementary material). KEGG analysis showed that 'metabolic parthways', 'biosynthesis of secondary metabolites' and 'phenylpropanoid' were significantly enriched (Fig. S3b, see online supplementary material). KOG analysis indicated that 'signal transduction mechanisms', 'secondary metabolites biosynthesis, transport, and catabolism', 'post-translational modification, protein turnover, chaperones' and were significantly enriched (Fig. S3c, see online supplementary material).
As shown in Fig. 2, among primary metabolite parthway biosynthesis enzyme genes, the expression of transcripts coding for glycolysis enzymes such as glyceraldehyde 3-phosphate dehydrogenase (SmGAPC) (SMil_00005750) was reduced.
In addition, two transcription factor genes involving in tanshinone and salvianolic acids regulation were detected by transcriptome profiling. SmERF6 (ethylene responsive factor) (SMil_00020272) was up-regulated and SmERF115 (SMil_00025335) was down-regulated.
To validate the transcriptome profiling results, 10 DEGs were chosen for qPCR analysis, including two transcription factor genes, 2 phenylpropanoid biosynthetic pathway genes, and 6 f lavonoid biosynthetic pathway genes (Fig. S4). Consistently, the qRT-PCR results follow the same trend as the transcriptome sequencing data.

SmMYB36 acts directly on SmGAPC, SmGGPPS1, SmCPS1, SmRAS, SmDXS2, SmERF6 and SmERF115 to synergistically regulate the accumulation of tanshinones and phenolic acids
To verify whether SmMYB36 acts directly on genes or regulators of tanshinone and phenolic acid biosynthesis, in vitro experiments EMSA were performed. As shown in Fig. 3a, SmMYB36 binds to the 3 × MBSI/II core motif probes in SmGAPC, SmRAS, SmDXS2, SmG-GPPS1, SmCPS1, SmERF6 and SmERF115, but not to the mutation probes.
To validate that SmMYB36 is acting as a regulator of target genes, in vivo experiments were performed with dual-LUC. SmGAPC, SmRAS, SmDXS2, SmGGPPS1, SmCPS1, SmERF6, and SmERF115 promoters drive the LUC gene to form a reporter, and SmMYB36 was overexpressed as an effector in the presence of the 35S promoter. LUC luminescence assays showed that co-expression with SmMYB36 increased the expression of SmDXS2pro::LUC, SmGGPPS1pro::LUC, SmCPS1pro::LUC, and SmERF6pro::LUC reporter genes, and declined the expression of SmGAPCpro::LUC, SmRASpro::LUC, and SmERF115pro::LUC expression, compared to controls lacking 35Spro::SmMYB36 (Fig. 3b). Further analysis of the mechanism of SmMYB36-SmERF6/SmERF115 co-regulation of SmCPS1 and SmRAS using dual-LUC assay also supported the finding that SmMYB36 and SmERF6 together enhanced the expression of SmCPS1, and SmMYB36 and SmERF115 acted antagonistically on the regulation of SmRAS ( Fig. 4a and b). Accordingly, these results suggest that SmMYB36 is a positive regulator of tanshinones biosynthesis and a negative regulator of phenolic acids synthesis.

The C-terminus of SmMYB36 has an acidic region that is required for transcriptional activation and activation of target genes
To identify the specific location of the transcriptional activation domain of SmMYB36, we constructed its truncated pGBKT7 bait vector. The truncated SmMYB36 containing the C-terminal acidic region SmMYB36 153-160 was transcriptionally active, and the rest (SmMYB 1-111 , SmMYB36 1-153 , SmMYB36 112-153 ) lost transcriptional activity (Fig. 5a).
To confirm whether the transcriptional activation domain of SmMYB36 is associated with the activation of target genes, SmMYB36 1-153 was constructed as the effector vector for dual-LUC experiments (Fig. 5b). It was found that compared with SmMYB36, SmMYB36 1-153 lost its ability to activate positively regulated target genes, but its effect on repressing target genes was still retained.
To further determine the function of amino acids at the Cterminus of SmMYB36 in S. miltiorrhiza endogenous, transgenic hairy roots of SmMYB36 (36O) and  and SmMYB36-SRDX transgenic hairy roots. c, d Expression levels of SmCPS1, SmKSL1, and SmCYP76AH1, the key enzyme genes of tanshinone synthesis branch, and SmPAL1, SmRAS, and SmCYP98A14, the key enzyme genes of salvianoli synthesis branch. Empty ATCC15834 of transgenic hairy roots was used as control and error bars indicate standard deviation (SD) of three biological replicates. As determined by analysis of one-way and Duncan's multiple range test (P < 0.05), different letters indicate significant differences. material), the color of the control and 36 T transgenic hairy roots and extracts was lighter compared to that of 36O. Three lines with 3-7-fold higher SmMYB36 expression levels than the controls (ATCC and EV) were selected separately for follow-up experiments (Fig. S6, see online supplementary material). The results of the HPLC assay showed that compared with the overexpression lines of SmMYB36 (36O-1, 36O-2, and 36O-3), the overexpression lines of SmMYB36 1-153 (36 T-1, 36 T −2, and 36 T-3) showed lower levels of tanshinones (DTI, CT, TA1, TAIIA, and TTA) and their biosynthesis-related target genes (SmDXS2, SmGGPPS1, SmCPS1, and SmERF6), while the levels of phenolic acids (RA, SalB, and TSA) and biosynthesis-related target genes (SmGAPC, SmRAS, and SmERF115) were largely unchanged (Fig. 6a-c). These results suggest that the acidic region at the C-terminus of SmMYB36 is responsible for the activating effect of the target gene, but is not related to its repressive function.

SmMYB36 acts as both activator and repressor
In this study, EMSA and Dual-luc assays revealed that SmMYB36 specifically binds to cis-acting elements of SmDXS2, SmGGPPS1, SmCPS1, and SmERF6 promoter regions and increases their expression levels, and also binds to specific DNA motifs of SmGAPC, SmRAS, and SmEFR115 but decreases their expression levels. These results suggest that SmMYB36 acts as both a transcriptional activator and a repressor. In truth, there are many studies that found that R2R3-MYB transcription factors mainly regulate the transcriptional levels of target genes as activators, with the majority of repressors belonging to subgroup 4 (SG4) [1,2]. It is highly conserved that the N-terminal end of R2R3-MYB TFs contains a functional DNA binding domain (MYB domain), while the C-terminal end possesses a variable activation or repression domain. Although we have identified that SmMYB36 has transactivation activity [32], the exact location and specific sequence of its activation domain are still unclear. Previously, it has been identified that functional MYB transcription factors such as GL1 (GLABRA1), WEREWOLF (WER), AtMYB23 and AtMYB2 contain a segment of acidic amino acid residues at their C-terminus that are required for transcriptional activation [36,37]. Similarly, an acidic amino acid residue (LELDEDE) was identified at the C-terminal 154 to 160 amino acids of SmMYB36, and we also demonstrated that it plays a decisive role in the transcriptional regulation of downstream target genes. Additionally, it is believed that the bHLH can interact with R2R3-MYB to enhance the transcriptional activation of R2R3-MYB [1]. SmMYB36 also contains a bHLH-interacting motif in the R3 structural domain ([D/E] Lx2[R/K]x3Lx6Lx3R), but the bHLH that interacts with it and the effect on its transcriptional activity has not been reported. The SG4 R2R3-MYB repressors is known to have a 'C2' repressor motif at the C-terminus containing an EAR motif core sequence (LXLXL), for which the mechanism of EAR action is not clear. In other repressor proteins, the EAR pattern functions by recruiting co-repressors [38]. It is puzzling that SmMYB36 does not belong to SG4 of the R2R3-MYB transcription factors and does not contain an EAR motif at the C-terminus, so it will be interesting to explore the mechanism of how it functions as a repressor. We speculate that SmMYB36 may have an unknown transcriptional repressor domain, and another possibility is that it can recruit other repressors to inhibit the transcription of target genes. For instance, two R2R3-MYB, GL1 (GLABRA1) and MYB75, belonging to the MBW complex, are able to interact with the recruitment of repressor JAZ proteins thereby inhibiting JA-mediated trichome initiation and anthocyanin accumulation [39]. The JAZ repressor also interacts with MYB21 and MYB24, which are required for stamen development, and represses their transcriptional activity [20]. The repression activity of AtMYB3 to phenylpropanoid biosynthesis enzyme cinnamate 4-hydroxylase (C4H) gene expression was enhanced via interaction with co-repressors AtLNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1) and AtLNK2 [40]. Therefore, the further studies could focus on identifying the transcriptional repression structural domains of SmMYB36 C-terminus, or focus on screening SmMYB36 interaction coactivators and co-repressors to deepen the mechanism of SAMMYB36 activators and repressors.

SmMYB36 coordinately regulates the primary and secondary metabolic pathway
Recently, large-scale yeast monohybrid experiments have demonstrated that most transcription factors in plants bind to the promoters of multiple metabolic pathway genes, instead of binding specifically to the promoter of a particular pathway gene. In general, different transcription factors can co-regulate different metabolic pathways [41]. It is well known that the products of plant primary metabolism provide energy and raw materials for secondary metabolism through glycolysis and tricarboxylic acid cycle (TAC). Previous studies have indicated that the metabolic f lux of phenylpropanoid biosynthesis is increased by overexpression of the 3-deoxy-D-arabinoheptulose 7-phosphate synthase (DAHPS) gene, which enhances the activity of the shikimate pathway [42,43]. Homoplastically, AtMYB12 not only activated the expression of enolase (ENO) in primary metabolic enzyme genes in glycolysis and DAHPS gene in the shikimate pathway, but also activated f lavonoid enzyme genes to drive carbon f lux toward phenylpropanoid biosynthesis [44]. Overexpression of SlMIXTA-like can increase phenylpropanoid content by binding to the promoter region of the gene encoding DAHPS, thereby increasing the f lux of downstream secondary metabolites [45]. Silencing of mitochondrial citrate synthase gene PhmCS or ATP-citrate lyase genes PaACLA1-A2 and PaACLB1-B2 reduced total anthocyanin content in petunia [46,47]. As a consequence, regulating the expression of primary metabolic genes is an effective way to alter the accumulation of secondary metabolites. In the present study, SmMYB36 regulated both SmGAPC in the primary metabolic glycolytic pathway and SmRAS gene in the secondary metabolic phenolic acid synthesis and SmDXS2, SmGGPPS1, and SmCPS1 genes in the tanshinone synthesis. In the glycolytic pathway, GAPC catalyzes the conversion of 3phosphoglycerate to 1,3-diphosphoglycerate. GAPC deficient mutant line shows a decrease in ATP levels and reduced levels of pyruvate and several TAC intermediates in Arabidopsis [48]. Of note, our results showed that down-regulation of SmGAPC by SmMYB36 results in less succinate in the Krebs cycle and reduced biosynthesis of phenylalanine and tyrosine, the precursors of salvianolic acids synthesis. This is detrimental to the availability of substances and energy for the synthesis of phenolic acids. In combination with the fact that SmMYB36 downregulated the expression of SmRAS, a key enzyme for phenolic acid synthesis, which further exacerbated the decrease in the accumulation of phenolic acid synthesis. Taken together, the down-regulation of phenolic acid accumulation in SmMYB36 overexpressing hairy roots is a result of the superimposed reduction in primary and secondary metabolism. Alternatively, for the increased accumulation of tanshinones in SmMYB36 overexpressing hairy roots was mainly attributed to the positive regulation of secondary metabolism key enzymes SmDXS2, SmGGPPS1, and SmCPS1 by SmMYB36, while the negative regulation of SmGAPC by SmMYB36 was clearly detrimental to its accumulation.

SmMYB36 coordinately directly and indirectly regulate tanshinone and salvianolic acid biosynthesis enzyme genes
SmMYB36 was shown to bind and activate SmERF6 and SmCPS1 promoters by EMSA and dual-LUC. Previous studies have demonstrated that SmERF6 binds to the GCC-box of the SmCPS1 promoter by EMSA and yeast one-hybrid (Y1H) and that it acts as an activator of tanshinone analog synthesis [22]. These results suggest that SmMYB36 positively regulates the accumulation of tanshinones directly or indirectly through SmERF6. We demonstrated by EMSA and dual-LUC that SmMYB36 binds to SmERF115 and SmRAS promoters to repress their expression. It has been previously demonstrated by yeast one-hybrid, EMSA and dual-LUC that SmERF115 binds and activates the GCC-box of the SmRAS promoter and that it acts as an activator of phenolic acid synthesis [21]. Thus, SmMYB36 negatively regulates the accumulation of phenolic acids directly or indirectly through SmERF115. Known as a feed-forward loop, this hierarchical regulatory network consists of one transcription factor regulating another transcription factor, which in turn co-regulates downstream targets [49]. Just as, in a multi-level feed-forward loop, MYB46/MYB83 coordinates with their regulators, secondary wall NACs (SWNs) proteins and their direct targets regulate a series of downstream target genes and secondary wall formation in Arabidopsis [50][51][52]. Particularly, this type of feed-forward loop regulatory mechanism has been reported in phenylpropanoid and terpenoid regulation. In the biosynthesis of artemisinin, AaMYC2 and AabZIP1 can be regulated by forming a bifurcated feedforward loop with TRICHOME-SPECIFIC WRKY 1 (AaGSW1), respectively [53]. The robustness of the signaling process can be improved by this coherent feed-forward loop of regulation [54]. In general, SmMYB36 further enhanced the positive and negative regulatory effects on tanshinones and phenolic acids through this feedforward loop regulation.

Mechanism of SmMYB36 positively and negatively regulating the synthesis of tanshinones and phenolic acids
Transcription factors regulating the synthesis of tanshinone and phenolic acid in S. miltiorrhiza have been reported extensively. SmMYC2a (SmMYC2), SmMYC2b and SmMYB98 as positive regulators and SmbHLH3 as negative regulators, while SmERF1L1, SmERF115, SmGRAS1, SmGRAS2 and SmbZIP1 are positive and negative bidirectional regulators [21,35,[55][56][57][58][59][60]. Studies on the mechanisms of regulation of tanshinone and phenolic acid by these transcription factors have focused on the regulation of key enzyme genes of the downstream synthetic pathway. Nonetheless, few studies have explored the mechanisms of primary metabolism level regulation and feed-forward loop regulation. Yang et al. (2017) [57] demonstrated by transcriptome that overexpression of SmMYC2 upregulated genes in the mangiferous acid synthesis pathway, but the necessary experimental evidence is lacking as to whether SmMYC2 directly initiates the expression of these genes and whether the upregulated expression of these genes enhances the accumulation of phenylalanine and tyrosine. Consequently, it cannot be ruled out that SmMYC2 has a direct up-regulatory effect on downstream genes of key enzymes for phenolic acid synthesis and forms a 'pull' on the upstream manganate pathway, rather than a simultaneous direct regulation of primary and secondary metabolic pathways by SmMYC2.   [59] showed that SmbZIP1 negatively regulated SmERF1L1, and the two formed a feed-forward loop to positively regulate the accumulation of tanshinones and phenolic acids. It can be understood that SmERF1L1 acts as a positive regulator of tanshinones and a negative regulator of phenolics, and SmbZIP1 inhibits the expression of phenolics negative regulators and thus promotes phenolics accumulation. It is puzzling that SmbZIP1 inhibited the positive regulator of tanshinones and the result of SmbZIP1 as the positive regulator of tanshinones is contrary. This suggests that there may be some complex regulatory mechanisms of SmbZIP1 as a positive regulator of tanshinones that have not been revealed. Hence, we found that SmMYB36 coordinates the regulation of tanshinone and phenolic acid through three levels: it acts as both activator and repressor in the first level; coordinates the regulation of primary and secondary metabolic pathways in the second level; and coordinates the regulation of tanshinone and phenolic acid biosynthetic enzyme genes directly and indirectly in the third level. Unexpectedly, the regulatory mechanisms for SmMYB36 specifically for the accumulation of tanshinone and phenolic acid are still remarkably different. SmMYB36 enhances tanshinone biosynthesis in S. miltiorrhiza through two mechanisms: (i) SmMYB36 initiated the transcription of SmDXS2, SmCPS1, and SmGGPPS1 to directly promote tanshinone biosynthesis; and (ii) SmMYB36 promotes the expression of SmERF6, the activator of tanshinone biosynthesis, and indirectly promotes tanshinone biosynthesis. Moreover, SmMYB36 decreased salvianolic acid biosynthesis in S. miltiorrhiza through three directions: (i) SmMYB36 directly down-regulated SmGAPC expression, which directly reduced accumulation of phenylalanine and tyrosine; (ii) SmMYB36 directly down-regulated SmRAS transcription abundance and thus directly inhibited phenolic acid synthesis; and (iii) SmMYB36 indirectly inhibited phenolic acid synthesis by suppressing SmERF115 expression, an activator of phenolic acid synthesis (Fig. 7).
In conclusion, the synthesis of phenolic acids was comprehensively inhibited by SmMYB36 from primary to secondary, and from direct to indirect; whereas the synthesis of tanshinones was positively regulated by SmMYB36 only for secondary metabolic pathways, both directly and indirectly. Our results fully demonstrate that SmMYB36 is functionally pleiotropic in regulating the synthesis of tanshinone and phenolic acid substances, one transcription factor acts on multiple pathways to exert multiple effects, which lays the foundation for a comprehensive understanding of the regulatory mechanism of the active ingredients of S. miltiorrhiza, and provides a useful tool for the future use of genetic engineering to improve the quality of S. miltiorrhiza and precisely regulate the active ingredients.

Plasmid construction
For constructing the hairy root genetic transformation vector, the complete open reading frame (ORF) sequence of SmMYB36 and its C-terminus containing SRDX (LDLDLELRLGFA) nucleotide sequence were cloned separately by PCR amplification and constructed onto the entry vector pDNOR207 using BP clonase enzyme. After that, the target sequences on the entry vector were constructed on pGWB18 and pK7WG2R using LR clonase enzyme to obtain pK7WG2R-SmMYB36-SRDX and pGWB18-Myc-SmMYB36, respectively. For transcriptional activation experiments, the amplified fragments (SmMYB36 1-160 , SmMYB36 1-111 , SmMYB36 1-153 , SmMYB36 112-160 , and SmMYB36 112-153 ) were constructed into the pDONR207 entry vector and pDEST-GBKT7 destination vector, separately and sequentially, using Gateway technology. For the functional characterization of SmMYB36 1-153 in transgenic hairy roots, the coding sequences (CDS) of SmMYB36 and SmMYB36 1-153 (LELDEDE with seven amino acids removed) were constructed into the pK7WG2R vector using Gateway technology. The above vector construction method was performed using gateway technology and the procedure follows the instructions of the BP and LR Clonase Kits (Invitrogen, USA).
For dual-LUC experiments, promoters of SmGAPC, SmRAS, SmDXS2, SmGGPPS1, SmCPS1, SmERF6, and SmERF115 were cloned and constructed into the pGreenII 0800-LUC vector as reporter vectors. The sequences of SmMYB36 and SmMYB36 1-153 were cloned into pCsGFPBT, and the full length of the ORFs of SmERF6 and SmERF115 were cloned into pGreenII 62-SK as effector vectors. The vector was constructed by a double digestion method according to the instructions of Clon Express MultiS One Step Cloning Kit (Vazyme Biotech, China). For EMSA assays, the  root. a, b The contents of DTI, CT, TAI, TAIIA and TTA, the major components of tanshinone, and RA, SaIB and TPA, the phenolic acids principal concentrations, in SmMYB36 and SmMYB36 1-153 transgenic hairy roots. c Expression levels of SmMYB36 target, genes SmGAPC, SmRAS, SmGGPPS1, SmCPS1, SmRAS, SmDXS2, SmERF6, and SmERF115. Empty ATCC15834 (ATCC) and empty vector (EV) of transgenic hairy roots was used as control and error bars indicate standard deviation (SD) of three biological replicates. A different letter indicates a significant difference between the groups, according to the analysis of one-way ANOVA and Duncan's multiple range test (P < 0.05).
CDS of SmMYB36 was constructed into the pET32a vector to obtain the fusion expression vector pET32a-His-SmMYB36. The gene sequence information involved in this study was obtained from the National Genome Data Center (https://ngdc.cncb.ac. cn/search/) under the accession number GWHAOSJ00000000. All primers involved in the above vector construction process are shown in Table S1 (see online supplementary material).

Plant transformation
The plasmids pGWB18-Myc-SmMYB36 (M36O), pK7WG2R-SmMYB36-SRDX (36R), pK7WG2R-SmMYB36 (36O), and pK7WG2R-SmMYB36 1-153 (36 T) were transformed with Agrobacterium rhizogenes ATCC15834 to induce S. miltiorrhiza hairy roots, ATCC15834 (ATCC) and transformants containing empty vector (EV) induced hairy roots and served as controls. Positive lines Figure 7. Schematic representation of SmMYB36 regulation of tanshinone and phenolic acid biosynthesis. The narrow solid arrows indicate single-step or proven reactions, while the wide dashed arrows represent multi-step or speculative reaction processes. Red font and arrows represent metabolites or genes that are positively regulated, blue arrows and font represent metabolites or genes that are negatively regulated.
of transgenic hairy roots were validated using the reported method [32].

Quantitative real-time PCR (qRT-PCR)
With the SteadyPure plant RNA extraction kit (ACCURATE, China), total RNA was extracted from transgenic hairy roots (M36O, 36R, 36O, and 36 T) and converted to cDNA with EvoM-MLV reverse transcription kit (ACCURATE). The qRT-PCR was performed following the previous method [32]. Three biological repetitions were conducted. All primers used in the qRT-PCR are found in Table S2 (see online supplementary material).

High-performance liquid chromatography (HPLC)
The dried hairy root samples of M36O, 36R, 36O, and 36 T transgenic and control lines were powdered with a mortar and pestle, weighed precisely 0.02 g in a 10 mL centrifuge tube, extracted with 4 mL of 70% methanol overnight and then treated with ultrasonic shaking for 45 min, centrifuged at 10000 rpm for 10 min at room temperature, the supernatant was removed and stored at 4 • C on a 0.45 μm filter membrane. As described previously, HPLC analysis was performed on three biological replicates of every sample [31]. Total phenolic acid (TPA) was determined by the sum of SalB (salvianolic acid B) and RA (rosmarinic acid), and the sum of tanshinone IIA (TIIA), while cryptotanshinone (CT), dihydrotanshinone I (DTI) and tanshinone I (TAI) was considered as total tanshinone (TTA).

Transcriptome and metabolome analysis
The widely targeted metabolome analysis and RNA sequencing were carried outby Wuhan Metware Biotechnology Co., Ltd (Metware Biotechnology, China). The analysis of metabolic pathways was perpformed by using the KEGG metabolic pathway database. A threshold for factors of variable importance in projection (VIP) ≥ 1 and fold change ≥2 or ≤ 0.5 was established for metabolites with significant differences in content. pGWB18-Myc-SmMYB36-transformed hairy roots were the experimental sample and ATCC15834-induced hairy roots were the control sample. Three replicates of each sample.
All clean reads were used with HISAT2 to map to the reference genome of S. miltiorrhiza [34]. Transcript abundance and differentially expressed genes (DEGs) were analysed identically to as previously described [33]. All DEGs were annotated according to GO; KEGG; KOG; Swiss-Prot; TrEMBL databases.

Electrophoretic mobility shift assays (EMSA)
HIS protein and recombinant protein His-SmMYB36 were purified as reported before [32]. Analysis of the 2000 bp promoter region upstream of the start codon ATG of SmGAPC, SmRAS, SmDXS2, SmGGPPS1, SmCPS1, SmERF6, and SmERF115 using the plantcare online site revealed core sequences containing MBSI and MBSII. Then, a 48 bp sequence containing the promoter fragment of the MBSI 3 × (CNGTT(G/A)) and 3 × MBSII (TNGTT(G/A)) core sequences was used as a probe, and the mutant MBSI 3 × (AAAAAA) and MBSII 3 × (AAAAAA) core sequences were used as control probes. The single-stranded probe was synthesized by Sangon Biotech, and only the biotin marker was added at the end of the 3 end of the sense strand, and no marker was added to the complementary strand. The solution was aspirated at F chain: R chain = 1:1, mixed well in the centrifuge tube, and placed in a PCR instrument for annealing at 90 • C for 10 min, and then slowly cooled down to room temperature to obtain a doublestranded probe master mix at a concentration of 10 μM. Follow-up experiments were performed according to the instructions of the LightShift EMSA Optimization and Control Kit (Thremo, China).

Dual-luciferase (dual-LUC) assays
Experiments with dual-LUC were conducted as previously described [35]. Different combinations of reporter and effector plasmids were co-transformed transiently into four-week-old tobacco (Nicotiana benthamiana) leaves. GloMax20/20 luminometers (Promega, USA) instrument and dual-Luciferase ® Reporter Assay System were used to determine the results. The results were performed in three biological replicates.

Data analysis
Data were processed with SPSS26.0 software, and one-way ANOVA and Duncan's multiple range test were used for significance analysis between three or more groups of samples, and Student's t-test was used for significance analysis between two groups of samples.